Amino-transfer transpeptidation

The pepsin-catalyzed transpeptidation reactions, both the amino transfer (14) and the acyl transfer types (15) (Fig.6), suggest the formation of at least two intermediates in which fragments of the substrate are bound to the enzyme. For a long time, however, no transpeptidation reaction of the amino transfer type could be found for substrates containing a blocked COOH-terminal carboxyl group adjacent to the bond being cleaved. The existence of the amino-enzyme in the pepsin hydrolysis pathway of conventional substrates was therefore somewhat doubtful (16). As for the transpeptidation of the acyl transfer type, it has been found so far only on substrates with a free NH2-terminal amino group. 

We have developed a technique for measuring initial formation rates of the products resulting from the amino transfer type of transpeptidation (17,18). To accomplish this, we used a chromophore acceptor which changed its spectral characteristics once an amide bond has been formed. The transpeptidation rate constant, k5, can be determined by analyzing the dependence of the initial rates on the acceptor concentration (Fig.7). 

In our studies, transpeptidation constants were compared for two substrate pairs containing the same amino acid residue to be transferred (Table IV). From this data, a common intermediate product, "amino-enzyme , was shown to exist for each substrate pair. Furthermore, the transpeptidation rate constants of substrates with a protected COOH-terminal amino acid were almost the same as that of substrates with a free carboxyl group. Difficulties encountered in detecting the transpeptidation product in the former case are due to an unfavorable ratio between the rates of hydrolysis and amino transfer. The effect of pH on the transpeptidation rate constants was studied (Fig.8) this constant was independent of pH in the pH range from 3.7 to 5.6. 

Figure 7. Kinetic Scheme for the reaction of transpeptidation of the amino transfer type, and theoretical dependence of the reciprocal transpeptidation rate on the reciprocal of the acceptor concentration.

One of the essential questions relating to the mechanism is whether the catalytic process involves any covalent intermediates between the substrate moieties and the enzyme. In the case of pepsin two possible intermediates could be formed the amino intermediate which involves the transfer of the amino moiety of the substrate to one of the carboxyl groups on the enzyme and the acyl intermediate which involves the transfer of the acyl moiety as shown on page 165. The examples used are substrates involved in transpeptidation reactions in which the respective intermediate has been demonstrated see below). 

Gln-Gln-Phe-Gly9-OMe was formed by chymotrypsin-catalyzed transpeptidation in the presence of H-Phe-GIy-OMe. In a papain-catalyzed acyl transfer reaction and subsequent tryptic cleavage, the resulting dodecapeptide ester was converted into substance P. These results indicate that peptides can be readily produced by a combination of recombinant DNA technology and peptidase-catalyzed conversion with the advantage of possible incorporation of groups other than coded amino acids into the recombinant product. 

During hydrolysis catalyzed by serine proteases an acyl-enzyme complex transfers the acyl group to water. However, in enzymatic synthesis, the acyl group is not transferred to water but to a nucleophile, that is, to an amino group or amino acids/peptides. Thus a transpeptidation reaction takes place during the enzymatic modification [46,57]. 

Transpeptidation, transamidation, a reaction involving the transfer of one or more amino acids from one peptide chain to another. This term was first coined by Fruton, in 1950, by analogy with transglycosidation for the papain-catalyzed displacement reaction between Bz-Gly-NH2 and aniline forming Bz-Gly-NHPh. Of special importance in relation to protease-catalyzed transpeptidation reactions in a preparative scale is the one-step tryptic conversion of porcine insulin into human insulin, despite the controversial interpretation of the mechanism involved. A bacterial transpeptidase, serim-type u-Ala-u-Ala carhoxypeptidase (EC... 

Transpeptidation In transpeptidation (peptidyl transfer), the peptide bond is formed via nucleophilic displacement of the P-site tRNA of the peptidyl-tRNA by the amino group of the aa-tRNA in the A-site. The nascent polypeptide chain is thereby lengthened at its C-terminus by one residue and transferred to the A-site tRNA (Figure 13.16). The peptidyl transfer reaction is catalyzed by the peptidyl transferase ribozyme of 23S rRNA of the 50S ribosome subunit. 

The enzyme works optimally at pH > 9 with N-protected amino acid esters as acyl donors, and amino acids or amino acid amides as nucleophiles (acceptors). At that pH value it has high esterase activity an amino acyl-enzyme complex will be formed rapidly which transfers the acyl residue to the acceptor molecule (transpeptidation), and since at alkaline pH the rate of peptide cleaving is minimal, the product formed will be released in excellent yield. As an example the last coupling step in a synthesis of the opioide peptide Met-enkephalin that started with benzoylarginine ethylester may be shown (Fig. 8). 

The enzymatic synthesis of peptides (Scheme 6.24) from which proteins can be constructed is not so limited, and chemical synthesis has an even wider application, but these are not yet suitable techniques for manufacture. Moreover, there are no general methods for building the peptides into full protein structures. Nevertheless, enzymes do have a role in the manufacture of peptides themselves. In a mixture of butan-l,4-diol and water, trypsin will catalyse the exchange of the carboxy-terminal alanine of porcine insulin with threonine t-butyl ester (Scheme 6.25). The reaction is essentially a transpeptidation in which the acyl group of lysine is transferred from one amino group on alanine to another on the threonine. This converts porcine insulin into the ester of the human hormone, and a simple deprotection yields one of the commercial products. 

All the known transpeptidation and transamidation reactions are of the carboxyl-transfer type and so far no example has been reported of the alternative type which might be envisaged, namely transfer of the amino moiety of a donor peptide to new linkage ivith the carboxyl group of an acceptor. 

My ne. t comments are concerned with the function of the enzymes responsible for the hydrolysis of GSH. We have felt from the start of our work in 1946 that the hydrolysis of GSH is concerned with absorption mechanisms and onlj" indirectly with the synthesis of protein. Thus, we would explain the transpeptidation reactions f rom GSH as a mechanism of transfer of amino acids across cellular membranes. The formation of 7-glutamyl peptides would provide the concentration differential necessary for the absorption of amino acids, for e.xample, from the tubular filtrate or from the intestine. Thus, we would explain the high concentration of the enzymes responsible for transpeptidation in kidney and intestine on the basis that these tissues are the ones most concerned with absorption phenomena. 

F. J. R. Hird It seems to me that one of the main functions of this 7-glutamjd transferase enzyme is that it provides employment for biochemists. Mr. P. H. Springell of Melbourne University has examined the properties of the 7-glutamyl transferase from sheep kidney in some detail. He finds that this enzyme transfers they-glutamyl group to water and to amino acids. The pH optimum for hydrolysis is about 6.0 and for transpeptidation about pH 8.5 or higher. He also finds that amino acids compete with water for the activated linkage and this competition is weaker at pH 6.0 than it is at pH 8.5. The amino acids which are the best reactors in transpeptidation are also the best competitors with water. 

The enzyme for reaction (a) was shown not to be the same as that for the synthesis of glutamine from glutamic acid and ammonia. Neither did the purified enzymes for reactions (a) and (b) contain the enzyme that hydrolyzes glutathione. Therefore both transpeptidation and a transfer reaction analogous to 7-glutamyl transamidation are excluded from the mechanism of the synthesis of glutathione from its amino acids. 

Our proposed mechanism is also compatible with the transpeptida-tion phenomenon which only occurs significantly for acid proteases at pH values greater than 4.0. Transfer of the proton from the carboxyl group of one hydrolysis product to the amino group of the other in Figure 8B could readily occur to produce R-COO" and H3 N-R. The transpeptidation products from the incubation of the peptide Leu-Try-Met with pepsin and penicillopepsin are the amino-trans-peptidation products Leu-Leu and Leu-Leu-Leu and the acyl-transpeptidation products Met-Met and Met-Met-Met (40). In order to produce the first products Leu-Leu and Leu-Leu-Leu, the tripeptide would bind initially such that the Leu-Trp bond is cleaved. Exchange of Trp-Met with a second Leu-Trp-Met is more facile than the exchange of the bound Leu product from the first hydrolysis. Transpep-... 

It has long been known that pepsin catalyses transpeptidation reactions with the apparent transfer of the amine product to an acidic acceptor, and this finding has been taken to indicate the prior departure of the acidic product and the formation of a covalently-bound amino-enzyme (3,13). Until recently, such transpeptidation reactions were observed largely with acyl dipeptides (e.g., Ac-Phe-Tyr) as substrates, and the corresponding esters or amides did not yield measurable amounts of transpeptidation product with H-labeled Ac-Phe as the acceptor (51). It has been reported, however, that Ac-Phe-Phe-y-propylmorpholinium ester does participate in a transpeptidation reaction with Z-Phe(N02) as the acceptor (9). 

If this should be the case, either apparent acyl transfer or amine transfer would be possible by a direct condensation of the preferentially retained product and an acceptor that can readily displace the product that leaves more easily. That such condensation reactions are catalyzed by pepsin in the case of oligopeptides was demonstrated many years ago (54), and is consistent with the neglible free energy decrease in the hydrolysis of interior peptide bonds (55). For transpeptidation reactions in which an apparent E-Tyr amino-enzyme has been postulated, the free energy change in the condensation of an acceptor such as Ac-Phe with tyrosine would be more unfavorable in free solution, but the possibility must be considered that the ammonium pKa of the tyrosine retained at the active site may be lower than that of tyrosine in free solution, perhaps by virtue of the interaction of the carboxylate group of the amino acid with a complementary cationic group of the active site. 

---